Kinesin inhibitors

ABSTRACT

The present invention provides for compounds, compositions, methods and systems for inhibiting cell growth. More specifically, the present invention provides for methods, compounds and compositions which are capable of inhibiting mitosis in metabolically active cells. Compounds, compositions and methods of the present invention inhibit the activity of a protein involved in the assembly and maintenance of the mitotic spindle. One class of proteins which acts on the mitotic spindle is the family of mitotic kinesins, a subset of the kinesin superfamily.

PRIORITY INFORMATION

The present application claims priority under 35 U.S.C. § 119(e) to theU.S. provisional patent application Ser. No. 60/184,540 by Mitchison etal. filed on Feb. 24, 2000. U.S. Ser. No. 60/184,540 is incorporatedherein by reference in its entirety.

GOVERNMENT SUPPORT

The work described in the present application was supported by a grantfrom the National Institutes of Health (CA78048).

BACKGROUND OF THE INVENTION

Cell-permeable small molecules can rapidly perturb the function of theirtargets and are therefore powerful tools to dissect dynamic cellularprocesses. However, such modulators are not available for most of theproteins involved in essential processes, and many of the ones that areavailable are nonspecific. The only known small molecules thatspecifically affect the mitotic machinery target tubulin (E. Hamel, Med.Res. Rev. 16, 207 (1996)), a subunit of the microtubules in the mitoticspindle.

One class of proteins involved in the assembly and maintenance of themitotic spindle is the family of mitotic kinesins, a subset of thekinesin superfamily. This superfamily contains over 100 proteins, whoseother functions include organelle transport and membrane organization(R. D. Vale and R. J. Fletterick, Annu. Rev. Cell Dev. Biol. 13, 745(1997)). The first evidence that mitotic kinesins are important inestablishing spindle bipolarity came from genetic studies:temperature-sensitive mutants in the BimC family of kinesins do not formbipolar spindles at the restrictive temperature (A. P. Enos and N. R.Morris, Cell 60, 1019 (1990); I. Hagan and M. Yanagida, Nature 356, 74(1992); M. A. Hoyt et al., J. Cell Biol. 118, 109 (1992)). Inhibition ofthe BimC kinesin Eg5 with Eg5-specific antibodies also inducedmonoasters similar to those observed after treatment with monastrol (A.Blangy et al., Cell 83, 1159 (1995); K. E. Sawin et al., Nature 359, 540(1992)). Like other kinesins, Eg5 can drive the movement of microtubulesin vitro (T. M. Kapoor and T. J. Mitchison, Proc. Natl. Acad. Sci.U.S.A. 96, 9106 (1999)).

Enzymes in the kinesin superfamily use the free energy of ATP hydrolysisto drive intracellular movement and influence cytoskeleton organization(R. D. Vale and R. J. Fletterick, Annu. Rev. Cell. Dev. Biol. 13,745-777 (1997)). More than 90 members of this family are known.Historically, kinesins have been proposed to move cellular cargo alongpolar microtubule tracks. More recently it has been shown that theseATPases can modulate dynamics of the underlying microtubule network (A.Desai et al., Cell 96, 69-78 (1999)), couple movement of cargo to themicrotubule polymerization or depolymerization (K. W. Wood et al., Cell91, 357-366 (1997)), and crosslink microtubules in dynamic structures(D. J. Sharp et al., J. Cell Biol. 144, 125-138 (1999)). Kinesins thusplay central roles in mitotic and meiotic spindle formation, chromosomealignment and separation, axonal transport, endocytosis, secretion, andmembrane trafficking. The cargo associated with these motor proteinsincludes intracellular vesicles, organelles, chromosomes, kinetochores,intermediate filaments, microtubules, and even other motors (reviewed inC. E. Walczak and T. J. Mitchison, Cell 85, 943-946 (1996); and N.Hirokawa, Science 279, 519-526 (1998)).

For many of these processes, more than one kinesin is implicated, andthe specific cargo associated with a given motor protein has beendifficult to establish. For example, conventional kinesin (R. D. Vale etal., Cell 42, 39-50 (1985)) (the founding member of the family) is oneof a subset of kinesins involved in organelle transport in mammaliancells. This group includes KIF1, KIF2, KIFC2/C3, and KIF4; and morerecently, 18 new murine KIFs have been reported, many of which mayfunctionally overlap with the transport kinesins (reviewed in N.Hirokawa, Science 279, 519-526 (1998)). It thus has been difficult totie down the in vivo function(s) of conventional kinesin. Experimentsusing antisense techniques and microinjection of inhibitory antibodieshave been further complicated by recent observations of efficientendoplasmic reticulum to Golgi transport in the absence of microtubules,albeit under restricted conditions (reviewed in G. S. Bloom and L. S.Goldstein, J. Cell Biol. 140, 1277-1280 (1998)). Similar problems havebeen encountered in dissecting the function of kinesins in mitosis.Extensive genetic analysis of motors in Saccharomyces cerevisiae haslinked all but one of the six kinesins to spindle function. None ofthese five motors are individually required for the viability of yeast,implying that more than one motor is associated with essential aspectsof spindle movement (W. S. Saunders and M. A. Hoyt, Cell 70, 451-458(1992); M. A. Hoyt et al., Proc. Natl. Acad Sci USA 94, 12747-12748(1997)). Immunodepletion and add-back approaches in Xenopus extractspindle assembly assays have provided similarly ambiguous data (C. E.Walczak et al., Curr. Biol. 8, 903-913 (1998)).

Small molecules that conditionally activate or inactivate a protein arevaluable tools for analyzing cellular functions of proteins (D. T. Hunget al., Chem. Biol. 3, 623-639 (1996)). Their use provides analternative to conventional biochemical and genetic approaches. However,to date there have been few reports of small molecules that canreversibly alter the function of motor proteins. Butanedione monoximehas been used to probe the role of myosin in cell movement (L. P. Cramerand T. J. Mitchison, J. Cell Biol. 131, 179-189 (1995)), but itsspecificity has been questioned (G. Steinberg and J. R. McIntosh, Eur.J. Cell Biol. 77, 284-293 (1998)). A natural product inhibitor ofkinesin has been reported (R. Sakowicz et al., Science 280, 292-295(1998)), but is thought not to be selective for different kinesins andthus is not useful for probing the role of one specific kinesin in acomplex process. Hyman et al. (A. A. Hyman et al., Nature (London) 359,533-536 (1992)) have used ATP analogs to distinguish between microtubulemotility at kinetochores driven by a kinesin and a dynein, but again,this approach is unlikely to distinguish between different kinesins.Thus currently there is a lack of small molecule activators orinhibitors that are specific for one member of the kinesin family. Suchan inhibitory molecule with specificity for a particular member of akinesin class would be useful as an anti-mitotic and also as ananti-cancer, anti-tumorigenic compound.

SUMMARY OF THE INVENTION

The present invention provides compounds, compositions, methods andsystems for inhibiting cell growth. More specifically, the presentinvention provides methods, compounds and compositions that are capableof inhibiting mitosis in metabolically active cells. Compounds, andcompositions of the present invention inhibit the activity of a proteininvolved in the assembly and maintenance of the mitotic spindle. Oneclass of proteins which acts on the mitotic spindle is the family ofmitotic kinesins, a subset of the kinesin superfamily.

In one aspect, the present invention provides a compound having theformula (I):

wherein R_(1-4, 8-10), as valency and stability permit are eachindependently selected from the group consisting of substitutedor-unsubstituted, branched or unbranched alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heterocycle,heteroalkyl, OH, OR_(A), C(═O)R_(A), CO₂H, CO₂R_(A), CN, halogen, SH,SR_(A), SOR_(A), SO₂R_(A), NO₂, NH₂, NHR_(A), N(R_(A))₂, hydrogen andNHC(O)R_(A), wherein each occurrence of R_(A) as valency and stabilitypermit is independently selected from the group consisting ofsubstituted or unsubstituted, branched or unbranched alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,heteroalkyl, heterocycle, alkoxy, aryloxy, alkylthio, arylthio,heteroaryloxy, heteroarylthio, and hydrogen, and

-   -   wherein R_(5-7, 11) as valency and stability permit are each        independently selected from the group consisting of substituted        or unsubstituted, branched or unbranched alkyl, alkenyl,        alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,        heteroaryl, heterocycle, heteroalkyl, hydrogen, —(C═O)R_(B), and        —(SO₂)R_(B), wherein each occurrence of R_(B) as valency and        stability permit is independently selected from the group        consisting of substituted or unsubstituted, branched or        unbranched alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,        cycloalkynyl, aryl, heteroaryl, heteroalkyl, heterocycle,        alkoxy, aryloxy, alkylthio, arylthio, heteroaryloxy,        heteroarylthio, hydrogen, and —(C═O)R_(C), wherein R_(C) as        valency and stability permit is selected from the group        consisting of substituted or unsubstituted, branched or        unbranched alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,        cycloalkynyl, aryl, heteroaryl, heteroalkyl, heterocycle,        alkoxy, aryloxy, alkylthio, arylthio, heteroaryloxy,        heteroarylthio, and hydrogen.

In another aspect, the present invention provides a compound having theformula (II):

wherein X is H, O, halogen, CF₃, or carbon; and wherein R and R′ asvalency and stability permit are each independently selected from thegroup consisting of substituted or unsubstituted, branched or unbranchedalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,heteroaryl, heterocycle, heteroalkyl, hydrogen, —(C═O)R_(D), and—(SO₂)R_(D), wherein each occurrence of R_(D) as valency and stabilitypermit is independently selected from the group consisting ofsubstituted or unsubstituted, branched or unbranched alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,heteroalkyl, heterocycle, alkoxy, aryloxy, alkylthio, arylthio,heteroaryloxy, heteroarylthio, hydrogen, and —(C═O)R_(E), wherein R_(E)as valency and stability permit is selected from the group consisting ofsubstituted or unsubstituted, branched or unbranched alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,heteroalkyl, heterocycle, alkoxy, aryloxy, alkylthio, arylthio,heteroaryloxy, heteroarylthio, and hydrogen.

In yet another aspect, the present invention provides a compound havingthe formula (III):

wherein R, R′ and R″ as as valency and stability permit are eachindependently selected from the group consisting of substituted orunsubstituted, branched or unbranched alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heterocycle,heteroalkyl, hydrogen, - (C═O)R_(F), and —(SO₂)R_(F), wherein eachoccurrence of R_(F) as valency and stability permit is independentlyselected from the group consisting of substituted or unsubstituted,branched or unbranched alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalkyl, heterocycle,alkoxy, aryloxy, alkylthio, arylthio, heteroaryloxy, heteroarylthio,hydrogen, and —(C═O)R_(G), wherein R_(G) as valency and stability permitis selected from the group consisting of substituted or unsubstituted,branched or unbranched alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalkyl, heterocycle,alkoxy, aryloxy, alkylthio, arylthio, heteroaryloxy, heteroarylthio, andhydrogen.

In yet another aspect, the present invention provides a compound havingthe formula (IV)

wherein Ar is phenyl, p-hydroxyphenyl, m-hydroxyphenyl, m-methoxyphenyl,m-halogenphenyl or m-fluorophenyl and R is as valency and stabilitypermit are each independently selected from the group consisting ofsubstituted or unsubstituted, branched or unbranched alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,heterocycle, heteroalkyl, hydrogen, —(C═O)R_(H), and —(SO₂)R_(H),wherein each occurrence of R_(H) as valency and stability permit isindependently selected from the group consisting of substituted orunsubstituted, branched or unbranched alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalkyl,heterocycle, alkoxy, aryloxy, alkylthio, arylthio, heteroaryloxy,heteroarylthio, hydrogen, and —(C═O)R_(I), wherein R_(I) as valency andstability permit is selected from the group consisting of substituted orunsubstituted, branched or unbranched alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalkyl,heterocycle, alkoxy, aryloxy, alkylthio, arylthio, heteroaryloxy,heteroarylthio, and hydrogen.

In still yet another aspect, the present invention provides apharmaceutical composition comprising one or more of the compounds(I-IV) as described by the present invention and further comprising apharmaceutically acceptable carrier.

In yet another aspect, the present invention provides a pharmaceuticalcomposition comprising a compound having the formula (V):

and further comprising a pharmaceutically acceptable carrier.

In yet another aspect, the present invention provides a method oftreating an individual with a cancerous growth comprising administeringto the individual a therapeutically effective and non-toxic dose of acomposition comprising a compound having the formula (V):

In yet another aspect, the present invention provides a method oftreating an individual with a cancerous growth comprising administeringto the individual a therapeutically effective and non-toxic dose of acomposition comprising one or more of the compounds I-IV as described bythe present invention.

DESCRIPTION OF DRAWINGS

FIG. 1 depicts the fragmentation of the Golgi and the formation ofmono-astral spindles by cells treated with 22C16.

FIG. 2 depicts the results of experiments examining the motilityactivity of full length Xenopus Eg5 with and without the beta-carboline,22C16. Also shown is the washout experiment demonstrating that 22C16acts reversibly to inhibit the motility of Eg5.

FIG. 3 is a photograph of an automated screening microscope, with aschematic of the steps in the screening process.

FIG. 4 is a schematic depiction of the protocol of the synchronousscreen for exocytosis. The BSC1 cells were mixed with media containingthe adenovirus with the VSVG-GFP construct and plated at 3,000cells/well (˜80% confluency) in a 384-well tray. After transduction, thecells were kept for 18 hours at 40° C. (the non-permissive temperature)leading to accumulation of VSVG-GFP in the ER. 50-100 nL of eachchemical (in DMSO) was diluted with 40 μl of media and transferred toassay wells, followed by incubation for another hour at 40° C. The cellswere then either (a) retained at 40° C. for 2 hours or incubated for 2hours at: (b) 20° C.; or (c) 32° C. With no inhibitors present, VSVG-GFPbehaves as follows: (a) retained in ER; (b) exits the ER but is retainedin the trans-Golgi network; (c) exits the ER, traffics to the Golgi andcontinues to the plasma membrane. Traffic was ended by transferring thetrays to 4° C. and fixing with 4% paraformaldehyde.

FIG. 5 shows the intracellular localization of VSVG-GFP along thesecretory pathway. These images demonstrate (a) the retention ofVSVG-GFP in the ER in cells kept at 40° C. The ER appears as a reticulumthroughout the cell. (b) Retention of VSVG-GFP in the perinuclearregions corresponding to the Golgi complex in cells incubated at 20° C.;and (c) Accumulation of VSVG-GFP at the plasma membrane, following exitfrom the ER and traffic through the Golgi in cells incubated at 32° C.

FIG. 6 shows images of cells with disruption in membrane traffic ofVSVG-GFP due to various hits. These images are typical examples of thetype of disruptions observed during the primary screen induced by thechemicals following the protocol summarized in FIG. 4. To facilitate thepresentation of the data, we only show ¼ of the complete field.

FIG. 7 depicts a description of the NADH enzyme coupled ATPase assayused to study the inhibition of the ability of purified humanrecombinant Eg5 to hydrolyze ATP in the presence and absence of 22C16and monastrol.

FIG. 8 depicts data from an experiment examining the inhibition of Eg5ATPase activity by the beta-carboline, 22C1 6. As indicated, the IC50for this experiment is 1.1 uM.

FIG. 9 depicts data from an experiment examining the inhibition of Eg5ATPase activity by monastrol. As indicated, the IC50 for this experimentis 5.3 uM.

FIG. 10 depicts the synthesis of monastroline.

FIG. 11 depicts the synthesis of derivatives of monastroline (Ar=phenyl,p-hydroxyphenyl, m-hydroxyphenyl, m-methoxyphenyl and m-fluorophenyl andR=hydrogen, methyl, ethyl, n-butyl, or α-benzyl). For conjugation ofmonastroline and derivatives to solid supports R can be C_(n)COOR, wheren=1-20, preferably 1, 3, 5, 7, 9, 11 or 13, and wherein R is methyl,ethyl, n-butyl or α-benzyl.

FIG. 12 depicts the synthesis of derivatives the beta-carboline core ofmonastroline starting with derivatives of tryptophan that are readilyavailable commercially.

FIG. 13 depicts the derivatives of monastroline where X is H, O Cl, Br,CF3, I or C.

FIG. 14 depicts the structure of a trans isomer of monastroline whichhas an IC50 approximately 8 times lower than for monastrol as assay bythe ATPase assay described in Example 2.

DEFINITIONS

As discussed above, the present invention provides a novel class ofcompounds useful for the treatment of cancer and other uncontrolled cellproliferative conditions related thereto. Compounds of this inventioncomprise those, as set forth above and described herein, and areillustrated in part by the various classes, subgenera and speciesdisclosed elsewhere herein.

“Therapeutically effective”: As used herein, the term “therapeuticallyeffective” is defined as an amount of a compound or compositioncomprising the compound which is administered to an individual in needthereof to slow or cease uncontrolled or abnormal growth of cells in theindividual without toxicity.

“Cancer or cancerous growth”: As used herein, the term “cancer” or“cancerous growth” means the uncontrolled, abnormal growth of cells andincludes within its scope all the well known diseases that are caused bythe uncontrolled and abnormal growth of cells. Non-limiting examples ofcommon cancers include bladder cancer, breast cancer, colon cancer,endometrial cancer, head and neck cancer, lung cancer, melanoma,non-hodgkin's lymphoma, prostate cancer, and rectal cancer. A completelist of cancers is available from the National Cancer Institute(Bethesda, Md.).

It will be appreciated by one of ordinary skill in the art that numerousasymmetric centers exist in the compounds of the present invention.Thus, inventive compounds and pharmaceutical compositions thereof may bein the form of an individual enantiomer, diastereomer or geometricisomer, or may be in the form of a mixture of stereoisomers.Additionally, in certain preferred embodiments, as detailed herein, themethod of the present invention provides for the stereoselectivesynthesis of alkaloids and analogues thereof. Thus, in certainembodiments, the compounds of the invention are enantiopure.

Additionally, the present invention provides pharmaceutically acceptablederivatives of the foregoing compounds, and methods of treating asubject using these compounds, pharmaceutical compositions thereof, oreither of these in combination with one or more additional therapeuticagents. The phrase, “pharmaceutically acceptable derivative”, as usedherein, denotes any pharmaceutically acceptable salt, ester, or salt ofsuch ester, of such compound, or any other adduct or derivative which,upon administration to a patient, is capable of providing (directly orindirectly) a compound as otherwise described herein, or a metabolite orresidue thereof. Pharmaceutically acceptable derivatives thus includeamong others pro-drugs. A pro-drug is a derivative of a compound,usually with significantly reduced pharmacological activity, whichcontains an additional moiety which is susceptible to removal in vivoyielding the parent molecule as the pharmacologically active species. Anexample of a pro-drug is an ester which is cleaved in vivo to yield acompound of interest. Pro-drugs of a variety of compounds, and materialsand methods for derivatizing the parent compounds to create thepro-drugs, are known and may be adapted to the present invention.Certain exemplary pharmaceutical compositions and pharmaceuticallyacceptable derivatives will be discussed in more detail herein below.

Certain compounds of the present invention, and definitions of specificfunctional groups are also described in more detail below. For purposesof this invention, the chemical elements are identified in accordancewith the Periodic Table of the Elements, CAS version, Handbook ofChemistry and Physics, 75^(th) Ed., inside cover, and specificfunctional groups are defined as described therein. Additionally,general principles of organic chemistry, as well as specific functionalmoieties and reactivity, are described in “Organic Chemistry”, ThomasSorrell, University Science Books, Sausalito: 1999, the entire contentsof which are incorporated herein by reference.

Furthermore, it will be appreciated by one of ordinary skill in the artthat the synthetic methods, as described herein, utilize a variety ofprotecting groups. By the term “protecting group”, has used herein, itis meant that a particular functional moiety, e.g., O, S, or N, istemporarily blocked so that a reaction can be carried out selectively atanother reactive site in a multifunctional compound. In preferredembodiments, a protecting group reacts selectively in good yield to givea protected substrate that is stable to the projected reactions; theprotecting group must be selectively removed in good yield by readilyavailable, preferably nontoxic reagents that do not attack the otherfunctional groups; the protecting group forms an easily separablederivative (more preferably without the generation of new stereogeniccenters); and the protecting group has a minimum of additionalfunctionality to avoid further sites of reaction. As detailed herein,oxygen, sulfur, nitrogen and carbon protecting groups may be utilized.Exemplary protecting groups are detailed herein, however, it will beappreciated that the present invention is not intended to be limited tothese protecting groups; rather, a variety of additional equivalentprotecting groups can be readily identified using the above criteria andutilized in the method of the present invention. Additionally, a varietyof protecting groups are described in “Protective Groups in OrganicSynthesis” Third Ed. Greene, T. W. and Wuts, P. G., Eds., John Wiley &Sons, New York: 1999, the entire contents of which are herebyincorporated by reference. Furthermore, a variety of carbon protectinggroups are described in Myers, A.; Kung, D. W.; Zhong, B.; Movassaghi,M.; Kwon, S. J. Am. Chem. Soc. 1999,121, 8401-8402, the entire contentsof which are hereby incorporated by reference.

It will be appreciated that the compounds, as described herein, may besubstituted with any number of substituents or functional moieties. Ingeneral, the term “substituted” whether preceded by the term“optionally” or not, and substituents contained in formulas of thisinvention, refer to the replacement of hydrogen radicals in a givenstructure with the radical of a specified substituent. When more thanone position in any given structure may be substituted with more thanone substituent selected from a specified group, the substituent may beeither the same or different at every position. As used herein, the term“substituted” is contemplated to include all permissible substituents oforganic compounds. In a broad aspect, the permissible substituentsinclude acyclic and cyclic, branched and unbranched, carbocyclic. andheterocyclic, aromatic and nonaromatic substituents of organiccompounds. For purposes of this invention, heteroatoms such as nitrogenmay have hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valencies of theheteroatoms. Furthermore, this invention is not intended to be limitedin any manner by the permissible substituents of organic compounds.Combinations of substituents and variables envisioned by this inventionare preferably those that result in the formation of stable compoundsuseful in the treatment of cancer and/or the inhibition of the growth ofor the killing of cancer cells. The term “stable”, as used herein,preferably refers to compounds which possess stability sufficient toallow manufacture and which maintain the integrity of the compound for asufficient period of time to be useful for the purposes detailed herein.

The term “aliphatic”, as used herein, includes both saturated andunsaturated, straight chain (i.e., unbranched), branched, cyclic, orpolycyclic aliphatic hydrocarbons, which are optionally substituted withone or more functional groups. As will be appreciated by one of ordinaryskill in the art, “aliphatic” is intended herein to include, but is notlimited to, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, andcycloalkynyl moieties. Thus, as used herein, the term “alkyl” includesboth straight, branched and cyclic alkyl groups. An analogous conventionapplies to other generic terms such as “alkenyl”, “alkynyl” and thelike. Furthermore, as used herein, the terms “alkyl”, “alkenyl”,“alkynyl” and the like encompass both substituted and unsubstitutedgroups.

Unless otherwise specified, alkyl and other aliphatic groups preferablycontain 1-6, or 1- 3, contiguous aliphatic carbon atoms. Illustrativealiphatic groups thus include, but are not limited to, for example,methyl, ethyl, n-propyl, isopropyl, cyclopropyl, —CH₂-cyclopropyl,allyl, n-butyl, sec-butyl, isobutyl, tert-butyl, cyclobutyl,—CH₂-cyclobutyl, n-pentyl, sec-pentyl, isopentyl, tert-pentyl,cyclopentyl, —CH₂-cyclopentyl, n-hexyl, sec-hexyl, cyclohexyl,—CH₂-cyclohexyl moieties and the like, which again, may bear one or moresubstituents. Alkenyl groups include, but are not limited to, forexample, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and thelike. Representative alkynyl groups include, but are not limited to,ethynyl, 2- propynyl (propargyl), 1-propynyl and the like.

The term “alkoxy”, or “thioalkyl” as used herein refers to an alkylgroup, as previously defined, attached to the parent molecular moietythrough an oxygen atom or through a sulfur atom. Examples of alkoxy,include but are not limited to, methoxy, ethoxy, propoxy, isopropoxy,n-butoxy, tert-butoxy, neopentoxy and n-hexoxy. Examples of thioalkylinclude, but are not limited to methylthio, ethylthio, propylthio,isopropylthio, n-butylthio, and the like.

The term “alkylamino” refers to a group having the structure —NHR′wherein R′ is alkyl, as defined herein. Examples of alkylamino include,but are not limited to, methylamino, ethylamino, iso-propylamino and thelike. In certain embodiments, C₁-C₃ alkylamino groups are utilized inthe present invention.

Some examples of substituents of the above-described aliphatic (andother) moieties of compounds of the invention include, but are notlimited to: F, Cl, Br, I, OH, NO₂, CN, C(O)-alkyl, C(O)-aryl,C(O)-heteroaryl, CO₂-alkyl, CO₂-aryl, CO₂-heteroaryl, CONH₂, CONH-alkyl,CONH-aryl, CONH-heteroaryl, OC(O)-alkyl, OC(O)-aryl, OC(O)-heteroaryl,OCO₂-alkyl, OCO₂-aryl, OCO₂-heteroaryl, OCONH₂, OCONH-alkyl, OCONH-aryl,OCONH-heteroaryl, NHC(O)-alkyl, NHC(O)-aryl, NHC(O)-heteroaryl,NHCO₂-alkyl, NHCO₂-aryl, NHCONH-heteroaryl, SO₂-alkyl, SO₂-aryl,C₃-C₆-cycloalkyl, CF₃, CH₂CF₃, CHCl₂, CH₂OH, CH₂CH₂OH, CH₂NH₂,CH₂SO₂CH₃, aryl, heteroaryl, benzyl, benzyloxy, aryloxy, heteroaryloxy,alkoxy, methoxymethoxy, methoxyethoxy, amino, benzylamino, arylamino,heteroarylamino, -alkyl-amino, thio, aryl-thio, heteroarylthio,benzyl-thio, alkyl-thio, or methylthiomethyl. Additional examples ofgenerally applicable substituents are illustrated by the specificembodiments shown in the Examples which are described herein.

In general, the terms “aryl” and “heteroaryl”, as used herein, refer tostable mono- or polycyclic, heterocyclic, polycyclic, andpolyheterocyclic unsaturated moieties having preferably 3-14 carbonatoms, each of which may be substituted or unsubstituted. Substituentsinclude, but are not limited to, any of the previously mentionedsubstitutents, i.e., the substituents recited for aliphatic moieties, orfor other moieties as disclosed herein, resulting in the formation of astable compound. In certain embodiments of the present invention, “aryl”refers to a mono- or bicyclic carbocyclic ring system having one or twoaromatic rings including, but not limited to, phenyl, naphthyl,tetrahydronaphthyl, indanyl, indenyl and the like. In certainembodiments of the present invention, the term “heteroaryl”, as usedherein, refers to a cyclic aromatic radical having from five to ten ringatoms of which one ring atom is selected from S, O and N; zero, one ortwo ring atoms are additional heteroatoms independently selected from S,O and N; and the remaining ring atoms are carbon, the radical beingjoined to the rest of the molecule via any of the ring atoms, such as,for example, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl,imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl,thiophenyl, furanyl, -quinolinyl, isoquinolinyl, and the like.

It will be appreciated that aryl and heteroaryl groups (includingbicyclic aryl groups) can be unsubstituted or substituted, whereinsubstitution includes replacement of one, two or three of the hydrogenatoms thereon independently with any one or more of the followingmoieties including, but not limited to: F, Cl, Br, I, OH, NO₂, CN,C(O)-alkyl, C(O)-aryl, C(O)-heteroaryl, CO₂-alkyl, CO₂-aryl,CO₂-heteroaryl, CONH₂, CONH-alkyl, CONH-aryl, CONH-heteroaryl,OC(O)-alkyl, OC(O)-aryl, OC(O)-heteroaryl, OCO₂-alkyl, OCO₂-aryl,OCO₂-heteroaryl, OCONH₂, OCONH-alkyl, OCONH-aryl, OCONH-heteroaryl,NHC(O)-alkyl, NHC(O)-aryl, NHC(O)-heteroaryl, NHCO₂-alkyl, NHCO₂-aryl,NHCONH-heteroaryl, SO₂-alkyl, SO₂-aryl, C₃-C₆-cycloalkyl, CF₃, CH₂CF₃,CHCl₂, CH₂OH, CH₂CH₂OH, CH₂NH₂, CH₂SO₂CH ₃, aryl, heteroaryl, benzyl,benzyloxy, aryloxy, heteroaryloxy, alkoxy, methoxymethoxy,methoxyethoxy, amino, benzylamino, arylamino, heteroarylamino,alkyl-amino, thio, aryl-thio, heteroarylthio, benzyl-thio, alkyl-thio,or methylthiomethyl. Additional examples of generally applicablesubstitutents are illustrated by the specific embodiments which aredescribed herein.

The term “cycloalkyl”, as used herein, refers specifically to groupshaving three to seven, preferably three to ten carbon atoms. Suitablecycloalkyls include, but are not limited to cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl and the like, which, as in the caseof other aliphatic, heteroaliphatic or hetercyclic moieties, mayoptionally be substituted. F, Cl, Br, I, OH, NO₂, CN, C(O)-alkyl,C(O)-aryl, C(O)-heteroaryl, CO₂-alkyl, CO₂-aryl, CO₂-heteroaryl, CONH₂,CONH-alkyl, CONH-aryl, CONH-heteroaryl, OC(O)-alkyl, OC(O)-aryl,OC(O)-heteroaryl, OCO₂-alkyl, OCO₂-aryl, OCO₂-heteroaryl, OCONH₂,OCONH-alkyl, OCONH-aryl, OCONH-heteroaryl, NHC(O)-alkyl, NHC(O)-aryl,NHC(O)-heteroaryl, NHCO₂-alkyl, NHCO₂-aryl, NHCONH-heteroaryl,SO₂-alkyl, SO₂-aryl, C₃-C₆-cycloalkyl, CF₃, CH₂CF₃, CHCl₂, CH₂OH,CH₂CH₂OH, CH₂NH₂, CH₂SO₂CH₃, aryl, heteroaryl, benzyl, benzyoxy,aryloxy, heteroaryloxy, alkoxy, methoxymethoxy, methoxyethoxy, amino,benzylamino, arylamino, heteroarylamino, alkyl-amino, thio, aryl-thio,heteroarylthio, benzyl-thio, alkyl-thio, or methylthiomethyl. Additionalexamples of generally applicable substitutents are illustrated by thespecific embodiments shown in the Examples which are described herein.

The term “heteroaliphatic”, as used herein, refers to aliphatic moietieswhich contain one or more oxygen, sulfur, nitrogen, phosphorous orsilicon atoms, e.g., in place of carbon atoms. Heteroaliphatic moietiesmay be branched, unbranched or cyclic and include saturated andunsaturated heterocycles such as morpholino, pyrrolidinyl, etc. Incertain embodiments, heteroaliphatic moieties are substituted byindependent replacement of one or more of the hydrogen atoms thereonwith one or more moieties including, but not limited to: F, Cl, Br, I,OH, NO₂, CN, C(O)-alkyl, C(O)-aryl, C(O)-heteroaryl, CO₂-alkyl,CO₂-aryl, CO₂-heteroaryl, CONH₂, CONH-alkyl, CONH-aryl, CONH-heteroaryl,OC(O)-alkyl, OC(O)-aryl, OC(O)-heteroaryl, OCO₂-alkyl, OCO₂-aryl,OCO₂-heteroaryl, OCONH₂, OCONH-alkyl, OCONH-aryl, OCONH-heteroaryl,NHC(O)-alkyl, NHC(O)-aryl, NHC(O)-heteroaryl, NHCO₂-alkyl, NHCO₂-aryl,NHCONH-heteroaryl, SO₂-alkyl, SO₂-aryl, C₃-C₆-cycloalkyl, CF₃, CH₂CF₃,CHCl₂, CH₂OH, CH₂CH₂OH, CH₂NH₂, CH₂SO₂CH₃, aryl, heteroaryl, benzyl,benzyloxy, aryloxy, heteroaryloxy, alkoxy, methoxymethoxy,methoxyethoxy, amino, benzylamino, arylamino, heteroarylamino,alkyl-amino, thio, aryl-thio, heteroarylthio, benzyl-thio, alkyl-thio,or methylthiomethyl. Additional examples of generally applicablesubstitutents are illustrated by the specific embodiments shown in theExamples which are described herein.

The terms “halo” and “halogen” as used herein refer to an atom selectedfrom fluorine, chlorine, bromine and iodine.

The term “haloalkyl” denotes an alkyl group, as defined above, havingone, two, or three halogen atoms attached thereto and is exemplified bysuch groups as chloromethyl, bromoethyl, trifluoromethyl, and the like.

The term “heterocycloalkyl” or “heterocycle”, as used herein, refers toa non-aromatic 5-, 6- or 7-membered ring or a bi- or tri-cyclic groupcomprising fused six-membered rings having between one and threeheteroatoms independently selected from oxygen, sulfur and nitrogen,wherein (i) each 5-membered ring has 0 to 1 double bonds and each6-membered ring has 0 to 2 double bonds, (ii) the nitrogen and sulfurheteroatoms may be optionally be oxidized, (iii) the nitrogen heteroatommay optionally be quaternized, and (iv) any of the above heterocyclicrings may be fused to a benzene ring. Representative heterocyclesinclude, but are not limited to, pyrrolidinyl, pyrazolinyl,pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl,oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl,isothiazolidinyl, and tetrahydrofuryl. In certain embodiments, a“substituted heterocycloalkyl or heterocycle” group is utilized and asused herein, refers to a heterocycloalkyl or heterocycle group, asdefined above, substituted by the independent replacement of one, two orthree of the hydrogen atoms thereon with but are not limited to: F, Cl,Br, I, OH, NO₂, CN, C(O)-alkyl, C(O)-aryl, C(O)-heteroaryl, CO₂-alkyl,CO₂-aryl, CO₂-heteroaryl, CONH₂, CONH-alkyl, CONH-aryl, CONH-heteroaryl,OC(O)-alkyl, OC(O)-aryl, OC(O)-heteroaryl, OCO₂-alkyl, OCO₂-aryl,OCO₂-heteroaryl, OCONH₂, OCONH-alkyl, OCONH-aryl, OCONH-heteroaryl,NHC(O)-alkyl, NHC(O)-aryl, NHC(O)-heteroaryl, NHCO₂-alkyl, NHCO₂-aryl,NHCONH-heteroaryl, SO₂-alkyl, SO₂-aryl, C₃-C₆-cycloalkyl, CF₃, CH₂CF₃,CHCl₂, CH₂OH, CH₂CH₂OH, CH₂NH₂, CH₂SO₂CH₃, aryl, heter benzyloxy,aryloxy, heteroaryloxy, alkoxy, methoxymethoxy, methoxyethoxy, amino,benzylamino, arylamino, heteroarylamino, alkyl-amino, thio, aryl-thio,heteroarylthio, benzyl-thio, alkyl-thio, or methylthiomethyl. Additionalexamples of generally applicable substitutents are illustrated by thespecific embodiments shown in the Examples which are described herein.

DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS

The present invention provides compounds, compositions, methods andsystems for inhibiting cell growth. More specifically, the presentinvention provides for methods, compounds and compositions which arecapable of inhibiting mitosis in metabolically active cells. Compounds,compositions and methods of the present invention inhibit the activityof a protein involved in the assembly and maintenance of the mitoticspindle. One class of proteins which acts on the mitotic spindle is thefamily of mitotic kinesins, a subset of the kinesin superfamily.

Monastrol

Mitchison and coworkers have demonstrated that thedihydropyrimidine-based compound monastrol is capable of arrestingmammalian cells in mitosis with monopolar spindles (Mayer et al. Science286:971-974, 1999; incorporated herein by reference). In vitro,monastrol specifically inhibited the motility of the mitotic kinesinEg5, a motor protein required for spindle bipolarity. Monastrol wasidentified as causing monoastral spindles in mitotic cells in amultistep screen. The initial screen utilized a whole-cellimmunodetection assay (Stockwell et al. Chem Biol February1999;6(2):71-83; WO 00/07017) to identify compounds that increased thephosphorylation of nucleolin. Nucleolin is a nucleolar protein that isspecifically phosphorylated in cells entering mitosis, and compoundsthat cause mitotic arrest would be expected to have increases levels ofphosphonucleolin. This initial screen identified 139 compounds from alibrary of 16,320 small molecules (Diverset E, Chembridge Corporation.San Diego, Calif.) as having anti-mitotic activity.

A secondary screen, utilizing an in vitro tubulin polymerization assay,ruled out molecules that target tubulin as its mode of action. Of the139 compounds selected in the first screen, 86 compounds did not arrestmitosis by targeting tubulin and were therefore deemed interesting forfurther study. These 86 compounds were then tested for their effects onmicrotubules, actin and chromosomes. Twenty-seven of the 86 compoundshad no observable effect on the microtubule and actin cytoskeleton or onchromosome distribution. Twelve of the 86 compounds had pleiotropiceffects and were not evaluated further. Forty-two of the 86 compoundsaffected cells in interphase as well as in mitosis and thus were notspecifically affecting mitosis. Cells treated with these small moleculeshad disorganized or partially depolymerized interphase microtubules inaddition to abnormal mitotic spindle structures and misalignedchromosomes.

Five of the 86 compounds altered the mitotic spindle specifically andwere thus studied further. Monastrol was identified from one of thesefive compounds. It was observed that monkey epithelial kidney cells(BS-C₇1) treated with monastrol had abnormal mitotic spindle. Thenormally bipolar mitotic spindle was replaced by a monoastralmicrotubule array surrounded by a ring of chromosomes. Interphase cellswere not affected.

In addition, by studying the effects of monastrol and a related compoundDHP2 on microtubule motility, Mayer et al. (Science 286:971-974, 1999)determined that the inhibition of monastrol on the Eg5 kinesin isspecific to monastrol. Furthermore, monastrol's inhibiting effect onmotility is specific for the Eg5 kinesin. Monastrol did not inhibitmicrotubule motility driven by conventional kinesin (Mayer et al.Science 286:971-974, 1999.)

Monastroline

It is an aspect of the present invention that a compound (andpharmaceutical compositions comprising the compound) having the formula(I) arrests cells in mitosis and therefore inhibits cell growth. Thecompound, a β-carboline class compound, is designated as 22C16 from theDiverset E library of small molecules (Chembridge Corporation) and isalso referred to herein as monastroline.

It is readily appreciated by one skilled in the art that the compounddesignated as 22C16 structure (depicted by the structural formula (V))contains two chiral centers and that the four stereoisomers of 22C16 areencompassed by the description of the present invention. Experiments onan optically purified stereoisomer showed that a trans isomer of 22C16shown in FIG. 14 has an IC50 which is approximately 8 times lower thanmonastrol.

The present invention teaches that monastroline affects the mitoticmachinery of mammalian cells by inhibiting the mitotic activity of akinesin. More specifically, 22C16 is capable of inhibiting the ATPaseactivity of the human mitotic kinesin Eg5. As previously described,enzymes in the kinesin family use the free energy of ATP hydrolysis todrive intracellular movement and effect cytoskeleton structure. Example2 describes experiments demonstrating that 22C16 inhibits the ATPaseactivity of a mitotic kinesin. These experiments utilized a purifiedrecombinant human kinesin, Eg5.

In addition, 22C16 is capable of arresting cells in mitosis. It wasobserved that 22C16 affected the spindles of mitotic mammalian cells ina manner similar to that observed for monastrol (Mayer et al. Science286:971-974, 1999). Monastrol causes the spindles in mitotic mammaliancells to form a mono-astral microtubule array surrounded by a ring ofchromosomes. This phenotype was also observed when 22C16 was added tomonkey-derived BSC1 cells (see Example 1 and FIG. 1).

In experiments studying the secretion of a viral G protein,approximately 140 compounds from the Diverset E library of smallmolecules (Chembridge Corp.) were found to affect the transport of atemperature sensitive mutant of a viral G protein fused to a greenfluorescent protein (GFP). More importantly, 42 of the 140 compoundswere found to affect the Golgi of the cells resulting in a fragmentedGolgi phenotype. During the analysis of these 42 compounds for theireffects on Golgi fragmentation and microtubule structure, it wasobserved that one compound, 22C16, fragmented the Golgi withoutaffecting interphase microtubules. In addition, 22C16 caused a spindledefect similar to that observed for monostral (FIG. 1). 22C16 causesspindles in mitotic mammalian cells to form a mono-astral microtubulearray surrounded by a ring of chromosomes.

When the effects of 22C16 and monastrol on microtubule structure weredirectly compared, it was shown by examining formation of mono-astralstructures that 22C16 has an IC50 (median inhibitory concentration) ofapproximately 2 uM as compared to the IC50 of approximately 20 uM formonastrol (Mayer and Mitchison, unpublished observations). Forexperimental details on the cell assay to determine mono-astralstructures, see Mayer et al. Science 286:971-974, 1999. Images of liveBS-C-l cells were taken as described (Cramer et al. Curr. Opin. Cell.Biol. 67:82, 1994). Briefly, for immunofluorescence, BS-C-1 cells werestained with DAPI (4′,6-diamidino-2-phenylindole; Sigma-Aldrich) tovisualize DNA and anti-α-tubulin (DM1 A; Sigma-Aldrich, St. Louis) tovisualize spindle structure.

Since the effect of 22C16 on mono-astral formation in mammalian cellswas similar to that of cells treated with monastrol, the ability of22C16 to inhibit kinesins was examined. Example 2 describes experimentsdemonstrating that 22C16 specifically affects the ability of the Eg5kinesin to hydrolyze ATP. It was shown that 22C16 inhibits the abilityof Eg5, but not conventional human kinesin, to hydrolyze ATP in thepresence of NADH and microtubules.

In addition, the ability of 22C16 to inhibit microtubule motility drivenby Eg5 was examined. For experimental protocols and details, see Mayeret al. Science 286:971-974, 1999. Full length Xenopus Eg5 was expressedin baculovirus according to standard protocols (see Coligan et al.Current Protocols in Protein Science and Ausubel et al. CurrentProtocols in Molecular Biology. John Wiley & Sons. Incorporated hereinby reference.) FIG. 2 shows that 22C16 reversibly inhibits microtubulemotility driven by full-length Eg5. For the washout, Eg5-drivenmicrotubule motility in the presence of 22C16 was measured. The assaychamber was then depleted of 22C16 and motility was immediately measuredagain. Therefore, the results of the experiment depicted in FIG. 2provide direct evidence that 22C16 inhibits the microtubule motilitydriven by Eg5.

Uses

Any system where the control of cellular growth and cell division isdesired may utilize compounds in the beta-carboline class to regulatemitosis. More specifically, in a preferred embodiment, the compoundpreviously designated and described as 22C16 may be used to inhibit cellgrowth. One non-limiting example of an application of 22C16 to acellular system is the use of 22C16 as an anti-mitotic anti-cancer drug.Other examples include controlling cell division and the immune systemin diseases such as rheumatoid arthritis.

In a preferred embodiment, a method of treating an individual withuncontrolled or abnormal cell growth is provided. Compositionscomprising monastroline or derivatives with similar biological activityare useful for treating individuals with cells that having becomecancerous tumors. As discussed monstroline and derivatives with similarstructure and biological activity are provided, preferably in aspharmaceutical composition containing a pharmaceutically acceptablecarrier. The compositions containing monstroline and/or derivatives canbe administered to an individual in need thereof at therapeuticallyeffective amounts to slow or cease the abnormal cell growth. Generally,abnormal cell growth is associated with cancerous cells. However, otherdiseases resulting from uncontrolled cell growth (e.g. cardiovasculardiseases, rheumatoid arthritis etc.) may be treated with compositionsand methods of the present invention.

Pharmaceutical Compositions

As discussed above, unexpectedly, the present invention provides novelcompounds having antitumor and anti-cell proliferative activity, andthus the inventive compounds are useful for the treatment of cancer.Accordingly, in another aspect of the present invention, pharmaceuticalcompositions are provided, wherein these compositions comprise any oneof the compounds as described herein, and optionally comprise apharmaceutically acceptable carrier.

In certain preferred embodiments, these compositions optionally furthercomprise one or more additional therapeutic agents. In certain otherembodiments, the additional therapeutic agent is an anticancer agent, asdiscussed in more detail herein. It will also be appreciated thatcertain of the compounds of present invention can exist in free form fortreatment, or where appropriate, as a pharmaceutically acceptablederivative thereof. According to the present invention, apharmaceutically acceptable derivative includes, but is not limited to,pharmaceutically acceptable salts, esters, salts of such esters, or anyother adduct or derivative which upon administration to a patient inneed is capable of providing, directly or indirectly, a compound asotherwise described herein, or a metabolite or residue thereof, e.g., aprodrug.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgement,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, S. M. Berge, etal. describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 66: 1-19 (1977), incorporated herein byreference. The salts can be prepared in situ during the final isolationand purification of the compounds of the invention, or separately byreacting the free base function with a suitable organic acid. Examplesof pharmaceutically acceptable, nontoxic acid addition salts are saltsof an amino group formed with inorganic acids such as hydrochloric acid,hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid orwith organic acids such as acetic acid, oxalic acid, maleic acid,tartaric acid, citric acid, succinic acid or malonic acid or by usingother methods used in the art such as ion exchange. Otherpharmaceutically acceptable salts include adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hernisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts,and the like. Representative alkali or alkaline earth metal saltsinclude sodium, lithium,. potassium, calcium, magnesium, and the like.Further pharmaceutically acceptable salts include, when appropriate,nontoxic ammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, loweralkyl sulfonate and aryl sulfonate.

Additionally, as used herein, the term “pharmaceutically acceptableester” refers to esters which hydrolyze in vivo and include those thatbreak down readily in the human body to leave the parent compound or asalt thereof. Suitable ester groups include, for example, those derivedfrom pharmaceutically acceptable aliphatic carboxylic acids,particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic acids, inwhich each alkyl or alkenyl moiety advantageously has not more than 6carbon atoms. Examples of particular esters includes formates, acetates,propionates, butyrates, acrylates and ethylsuccinates.

Furthermore, the term “pharmaceutically acceptable prodrugs” as usedherein refers to those prodrugs of the compounds of the presentinvention which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswith undue toxicity, irritation, allergic response, and the like,commensurate with a reasonable benefit/risk ratio, and effective fortheir intended use, as well as the zwitterionic forms, where possible,of the compounds of the invention. The term “prodrug” refers tocompounds that are rapidly transformed in vivo to yield the parentcompound of the above formula, for example by hydrolysis in blood. Athorough discussion is provided in T. Higuchi and V. Stella, Pro-drugsas Novel Delivery Systems, Vol. 14 of the A.C.S. Symposium Series, andin Edward B. Roche, ed., Bioreversible Carriers in Drug Design, AmericanPharmaceutical Association and Pergamon Press, 1987, both of which areincorporated herein by reference.

As described above, the pharmaceutical compositions of the presentinvention additionally comprise a pharmaceutically acceptable carrier,which, as used herein, includes any and all solvents, diluents, or otherliquid vehicle, dispersion or suspension aids, surface active agents,isotonic agents, thickening or emulsifying agents, preservatives, solidbinders, lubricants and the like, as suited to the particular dosageform desired. Remington's Pharmaceutical Sciences, Fifteenth Edition, E.W. Martin (Mack Publishing Co., Easton, Pa., 1975) discloses variouscarriers used in formulating pharmaceutical compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional carrier medium is incompatible with the anti-viralcompounds of the invention, such as by producing any undesirablebiological effect or otherwise interacting in a deleterious manner withany other component(s) of the pharmaceutical composition, its use iscontemplated to be within the scope of this invention. Some examples ofmaterials which can serve as pharmaceutically acceptable carriersinclude, but are not limited to, sugars such as lactose, glucose andsucrose; starches such as corn starch and potato starch; cellulose andits derivatives such as sodium carboxymethyl cellulose, ethyl celluloseand cellulose acetate; powdered tragacanth; malt; gelatin; talc;excipients such as cocoa butter and suppository waxes; oils such aspeanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; cornoil and soybean oil; glycols; such a propylene glycol; esters such asethyl oleate and ethyl laurate; agar; buffering agents such as magnesiumhydroxide and aluminum hydroxide; alginic acid; pyrogen-free water;isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffersolutions, as well as other non-toxic compatible lubricants such assodium lauryl sulfate and magnesium stearate, as well as coloringagents, releasing agents, coating agents, sweetening, flavoring andperfuming agents, preservatives and antioxidants can also be present inthe composition, according to the judgment of the formulator.

In yet another aspect, according to the methods of treatment of thepresent invention, tumor cells are killed, or their growth is inhibitedby contacting said tumor cells with an inventive compound orcomposition, as described herein. Thus, in still another aspect of theinvention, a method for the treatment of cancer is provided comprisingadministering a therapeutically effective amount of an inventivecompound, or a pharmaceutical composition comprising an inventivecompound to a subject in need thereof, in such amounts and for such timeas is necessary to achieve the desired result.

In certain embodiments of the present invention a “therapeuticallyeffective amount” of the inventive compound or pharmaceuticalcomposition is that amount effective for killing or inhibiting thegrowth of tumor cells. The compounds and compositions, according to themethod of the present invention, may be administered using any amountand any route of administration effective for killing or inhibiting thegrowth of tumor cells. Thus, the expression “amount effective to kill orinhibit the growth of tumor cells”, as used herein, refers to asufficient amount of agent to kill or inhibit the growth of tumor cells.The exact amount required will vary from subject to subject, dependingon the species, age, and general condition of the subject, the severityof the infection, the particular anticancer agent, its mode ofadministration, and the like.

The anticancer compounds of the invention are preferably formulated indosage unit form for ease of administration and uniformity of dosage.The expression “dosage unit form” as used herein refers to a physicallydiscrete unit of anticancer agent appropriate for the patient to betreated. It will be understood, however, that the total daily usage ofthe compounds and compositions of the present invention will be decidedby the attending physician within the scope of sound medical judgment.The specific therapeutically effective dose level for any particularpatient or organism will depend upon a variety of factors including thedisorder being treated and the severity of the disorder; the activity ofthe specific compound employed; the specific composition employed; theage, body weight, general health, sex and diet of the patient; the timeof administration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed; andlike factors well known in the medical arts.

Furthermore, after formulation with an appropriate pharmaceuticallyacceptable carrier in a desired dosage, the pharmaceutical compositionsof this invention can be administered to humans and other animalsorally, rectally, parenterally, intracisternally, intravaginally,intraperitoneally, topically (as by powders, ointments, or drops),bucally, as an oral or nasal spray, or the like, depending on theseverity of the infection being treated. In certain embodiments, thecompounds of the invention may be administered orally or parenterally atdosage levels of about 0.01 mg/kg to about 50 mg/kg and preferably fromabout 1 mg/kg to about 25 mg/kg, of subject body weight per day, one ormore times a day, to obtain the desired therapeutic effect.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Besides inert diluents,the oral compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of the drug from subcutaneous or intramuscular injection.This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle. Injectable depot forms are made by forming microencapsulematrices of the drug in biodegradable polymers such aspolylactide-polyglycolide. Depending upon the ratio of drug to polymerand the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar—agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand-bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes. Solid compositions of asimilar type may also be employed as fillers in soft and hard-filledgelatin capsules using such excipients as lactose or milk sugar as wellas high molecular weight polethylene glycols and the like.

The active compounds can also be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositionswhich can be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, ear drops, and eye drops are also contemplatedas being within the scope of this invention. Additionally, the presentinvention contemplates the use of transdermal patches, which have theadded advantage of providing controlled delivery of a compound to thebody. Such dosage forms can be made by dissolving or dispensing thecompound in the proper medium. Absorption enhancers can also be used toincrease the flux of the compound across the skin. The rate can becontrolled by either providing a rate controlling membrane or bydispersing the compound in a polymer matrix or gel.

As discussed above, the compounds of the present invention are useful asanticancer agents, and thus may be useful in the treatment of cancer, byeffecting tumor cell death or inhibiting the growth of tumor cells. Ingeneral, the inventive anticancer agents are useful in the treatment ofcancers and other proliferative disorders, including, but not limited tobreast cancer, cervical cancer, colon and rectal cancer, leukemia, lungcancer, melanoma, multiple myeloma, non-Hodgkin's lymphoma, ovariancancer, pancreatic cancer, prostate cancer, and gastric cancer, to namea few. In certain embodiments, the inventive anticancer agents areactive against leukemia cells and melanoma cells, and thus are usefulfor the treatment of leukemias (e.g., myeloid, lymphocytic, myelocyticand lymphoblastic leukemias) and malignant melanomas. In still otherembodiments, the inventive anticancer agents are active against solidtumors and also kill and/or inhibit the growth of multidrug resistantcells (MDR cells).

It will also be appreciated that the compounds and pharmaceuticalcompositions of the present invention can be employed in combinationtherapies, that is, the compounds and pharmaceutical compositions can beadministered concurrently with, prior to, or subsequent to, one or moreother desired therapeutics or medical procedures. The particularcombination of therapies (therapeutics or procedures) to employ in acombination regimen will take into account compatibility of the desiredtherapeutics and/or procedures and the desired therapeutic effect to beachieved. It will also be appreciated that the therapies employed mayachieve a desired effect for the same disorder (for example, aninventive compound may be administered concurrently with anotheranticancer-agent), or they may achieve different effects (e.g., controlof any adverse effects).

For example, other therapies or anticancer agents that may be used incombination with the inventive anticancer agents of the presentinvention include surgery, radiotherapy (in but a few examples,y-radiation, neutron beam radiotherapy, electron beam radiotherapy,proton therapy, brachytherapy, and systemic radioactive isotopes, toname a few), endocrine therapy, biologic response modifiers(interferons, interleukins, and tumor necrosis factor (TNF) to name afew), hyperthermia and cryotherapy, agents to attenuate any adverseeffects (e.g., antiemetics), and other approved chemotherapeutic drugs,including, but not limited to, alkylating drugs (mechlorethamine,chlorambucil, Cyclophosphamide, Melphalan, Ifosfamide), antimetabolites(Methotrexate), purine antagonists and pyrimidine antagonists(6-Mercaptopurine, 5-Fluorouracil, Cytarabile, Gemcitabine), spindlepoisons (Vinblastine, Vincristine, Vinorelbine, Paclitaxel),podophyllotoxins (Etoposide, Irinotecan, Topotecan), antibiotics(Doxorubicin, Bleomycin, Mitomycin), nitrosoureas (Carmustine,Lomustine), inorganic ions (Cisplatin, Carboplatin), enzymes(Asparaginase), and hormones (Tamoxifen, Leuprolide, Flutamide, andMegestrol), to name a few. For a more comprehensive discussion ofupdated cancer therapies see, http://www.nci.nih.gov/, a list of the FDAapproved oncology drugs athttp://www.fda.gov/cder/cancer/druglistframe.htm, and The Merck Manual,Seventeenth Ed. 1999, the entire contents of which are herebyincorporated by reference.

In still another aspect, the present invention also provides apharmaceutical pack or kit comprising one or more containers filled withone or more of the ingredients of the pharmaceutical compositions of theinvention, and in certain embodiments, includes an additional approvedtherapeutic agent for use as a combination therapy. Optionallyassociated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceutical products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration.

Equivalents

The representative examples which follow are intended to help illustratethe invention, and are not intended to, nor should they be construed to,limit the scope of the-invention. Indeed, various modifications of theinvention and many further embodiments thereof, in addition to thoseshown and described herein, will become apparent to those skilled in theart from the full contents of this document, including the exampleswhich follow and the references to the scientific and patent literaturecited herein. It should further be appreciated that the contents ofthose cited references are incorporated herein by reference to helpillustrate the state of the art. The following examples containimportant additional information, exemplification and guidance which canbe adapted to the practice of this invention in its various embodimentsand the equivalents thereof.

EXAMPLE 1 Screen to Identify Small Molecule Inhibitors of ExocytosisAutomated Image Capture

Automated screening microscope was developed to allow medium-throughputimaging-based screening. A Nikon inverted fluorescence microscopeequipped with a 20×dry lens (F 0.45) was equipped with a cooled chargedcoupled device (CCD) camera and the Metamorph software suite (UniversalImaging Corp.) for data acquisition. Changes in the focal plane wereobtained by controlling the focal length of the optical path with apiezo-electric collar attached to the 20× dry lens with steps of 5 μm ona total range of 300 μm. Best focus was achieved by using an algorithmthat searches for maximum contrast on the acquired image. The microscopeset-up is diagrammed in FIG. 3.

The Metamorph software performs the following tasks:

-   -   (1) automatically center the lens in each well of a tissue        culture plate (in our case, 384-well plates);    -   (2) automatically focus the image, by collecting 25 images in        different focal planes and identifying the image with maximum        contrast; and    -   (3) transfer the final in-focus image (642 kilobytes) to the        hard drive of the attached computer (Pentium III, 1 Gigabyte        RAM, 28 Gigabyte hard drive).

On average, data were collected from 20-40 cells per field. Completedata acquisition for a 384-well tray was accomplished in approximatelyone hour. The typical memory requirement for a screen of 10,000compounds is approximately 6 Gigabytes.

Implementation of the Synchronous Screen for Exocytosis

VSVG-ts045 (Gallione and Rose, J. Virology 54:374-82. 1985) is atemperature sensitive mutant of the G protein of VSV (vesicularstomatitis virus) that is retained in the ER if the cells are grown atthe non-permissive temperature of 40° C. When cells are transferred tothe permissive temperature of 32° C., VSVG-ts045 exits the ER andcontinues its traffic through the Golgi complex, finally reaching theplasma membrane. Others have shown that it is possible to add the greenfluorescence protein (GFP) to the cytoplasmic tail of VSVG-ts045(abbreviated here as VSVG-GFP), and that this chimera traffics in asimilar way (Hirschberg et al., J. Cell Biol. 143:1485-1503. 1998).

We generated a construct of VSVG-GFP and inserted it into anadenovirus-based expression vector (He et al., Proc. Natl. Acad. Sci.USA 95:2509-2514. 1998). The adenovirus containing the construct wasamplified in COS cells and aliquots of the media were used fortransduction in monkey-derived BSC1 cells. Almost 100% of the cellsexpress high amounts of VSVG-GFP that can be detected by fluorescencemicroscopy 18 hours after infection. The protocol used in the screen forexocytosis is shown in FIG. 4; an example of the type of images that areacquired in the absence of added compounds is shown in FIG. 5.

The images were retrieved using Metamorph, and scored for variations inthe pattern of VSVG-GFP traffic by direct visual inspection. Images wereclassified into one of several phenotypes, as shown in FIG. 6.

Summary of the Exocytic Screen

We tested the effect of ˜10,000 compounds (out of a total of 16,320)from the Chembridge collection (Diverset E; Chembridge Corporation) inthe traffic of VSVG-GFP following the protocol described above. About140 hits were founds with obvious effects on single steps in the trafficproperties of VSVG-GFP (FIG. 6). Twenty-six hits that act at nominalconcentrations of 100 μM or lower (based on dilutions of stocksolutions) were selected for further study. A summary of this stage ofthe screen is given in Table 1.

We next used a suite of secondary screens to explore how extensive theeffects of these chemicals are on treated cells. This secondary screenconsisted of tests for (a) perturbations in the intracellularorganization of the tubulin-based cytoskeleton; (b) variations in theintracellular distribution of ER, Golgi and lysosomal markers; and (c)changes in the rate of uptake and intracellular targeting to endosomesof Texas Red transferrin internalized by receptor-mediated endocytosis.

It was observed in the primary screen described that one compound 22C16fragmented the Golgi of treated mammalian cells. However, 22C16 did notdepolymerize interphase microtubules and in fact, caused the formationof monopolar spindles in mitotic cells. FIG. 1 shows the effects of22C16 on Golgi fragmentation and spindle structure. As viewed in thelower right panel of FIG. 1, 22C16 causes the formation of monospindleswith a mono-astral microtubule array surrounded by a ring ofchromosomes.

The observation of a mono-astral phenotype in mitotic cells treated with22C16 led to experiments to study the effects of 22C16 on Eg5 as waspreviously performed with monastrol (Mayer et al. Science 286:971-974,1999). The motility assay of 22C16 on Eg5 was described in the preferredembodiments of this application and the data are depicted in FIG. 2. Thedirect comparison of 22C16 and monastrol on the monopolar spindlemono-astral formation on mammalians was also described in the preferredembodiments.

EXAMPLE 2 ATPase Assay with Purified Human Eg5 Kinesin

Monastrol and 22C16 were both determined to block the ATPase activity ofhuman Eg5 (N-terminal 405 amino acids and 6-His tagged at C terminus).Monastrol did not inhibit the activity of human kinesin (N-terminal 560amino acids of full length protein followed by 6-His tag at C terminus).

Methods

Preparation of Recombinant Human Eg5 Kinesin (Eg5-405)

DNA encoding full length human Eg5 kinesin was amplified by thepolymerase chain reaction (PCR) using Vent DNA polymerase (NE Biolabs,Beverly, Mass.) and subcloned into an expression plasmid (pRSETa). Forthe PCR reaction, the template used was a pBluescript vector containingthe full length coding sequence for human Eg5 (a gift from Anne Blangy).The 5′ primer (5′-GCA ACG ATT AAT ATG GCG TCG CAG CCA AAT TCG TCT GCGAAG) contained an Ase I cleavage site upstream of the Eg5 start codon.The 3′ primer (5′-GCA ACG CTC GAG TCA GTG ATG ATG GTG GTG ATG CAT GACTCT AAA ATT TTC TTC AGA AAT ) was complimentary to amino acid 405 andadded a downstream six histidine tag (6-HIS) followed by a UGA stopcodon, and Xho I cleavage site.

The resulting PCR DNA amplification product and also the target plasmidto be used as the expression plasmid (pRSETa) were double digested withAse I/Xho I and Nde I/Xho I respectively (New England Biolabs). Bothproducts of the two restriction enzyme double digests were resolved andpurified by agarose gel electrophoresis. The bands on the agarose gelcorresponding to the desired DNA fragments, more than 2 kb for pRSETaand 1.2 kb for Eg5-405 were excised and purified (Qiagen GelPurification Kit). The cleaved and purified DNA fragments were ligatedtogether using T4 DNA ligase (New England Biolabs). The ligationproducts were transformed into E. coli DH5α chemically competent cells(Life Technologies), and selected by overnight growth on LB ampicillinplates. Transformants were amplified by growth of E. coli in LBampicillin. Plasmids were purified (Qiagen Midiprep), and sequenced(Harvard Medical School Biopolymer Facilities).

Purification of Eg5-405 and K560 (560 Amino Acid Kinesin Construct):

BL21 pLysS (DE3) bacteria were transfected with the expression plasmiddescribed in the preceding section and grown overnight at 37° C. on LBplates containing 100 ug/ml ampicillin (LB-amp). Several colonies werepicked and grown at 37° C. in 1 ml LB-amp, pooled and used to inoculateeach of six 1.5 L of LB-amp. These I L cultures were incubated at 37° C.on a shaker (200 RPM) until the optical density (O.D.) of the culturereached an absorbance of approximately A_(600 nM)=0.5 O.D. The cultureswere cooled to 20° C., induced with 24 mg/ml of isopropylbeta-D-thiogalactopyranoside (IPTG; Boehringer Mannheim) and incubatedat room temperature for approximately 3 hours. Cells were pelleted bycentrifugation (4000×g), rinsed in phosphate buffered saline (PBS) andrepelleted at 10,000×g). The bacterial pellets were flash frozen inliquid nitrogen and stored at −80° C.

The bacterial pellets were thawed on ice and resuspended in a solutioncontaining 50 mM potassium phosphate (pH 8.0), 250 mM KCl, 0.1%Tween-20, 10 mM imidazole, 0.5 mM magnesium adenosine triphosphate(Mg-ATP), 1 mM phenylmethanesulfonyl fluoride (PMSF), and 2 mMbenzimidine-HCl. To lyse the bacteria, lysozyme (1 mg/ml) and2-mercaptoethanol (5 mM) were added to the solution to result in theindicated final concentration, incubated and sonicated (3 times 20seconds, repeated 3 times incubating for 1 minute on ice between eachtriple sonication) to break up the DNA and guarantee bacterial lysis.

The lysate was spun at 40,000×g for approximately 35 minutes at 4° C.with the resulting supernatant separated from the pellet by decantingand then incubated with a nickel-nitrilotriacetic acid resin (Ni-NTA;QIAGEN). The remaining pellet was washed three times with a wash buffer(lysis buffer supplemented with 10 mM 2-mercaptoethanol, no PMSF, and0.1 mM Mg-ATP) to extract any remaining protein. These washes were thenfollowed by a final wash using a low pH buffer (pH 6.0). The washsolutions were then also added to the Ni-NTA resin. The resin containingthe desired tagged protein was poured into a column (Biorad, 0.8×4 cmPolyPrep Chromatography Column) and allowed to settle. The HIS-taggedproteins were eluted with a solution containing 250 mM imidazole and 150mM KCl (pH 7.0). Protein-containing fractions of the eluate were loadedonto a Superose 6 size-exclusion column (Pharmacia) and equilibratedwith a solution containing 80 mM potassium HEPES (pH 6.8), 200 mM KCl,10 uM Mg-ATP, 1 mM dithiothreitol (DTT). Fractions containinghomogeneous proteins as determined by molecular weight and mobility onSDS-PAGE were used for further enzymology experiments.

Polymerization of Microtubules:

A solution containing 1 mg/ml tubulin, 1 mM DTT, 1 mM guanosinetriphosphate (GTP), 1 mM MgCl₂, 80 mM potassium HEPES (pH 6.8), and 1 mMethylene glycol-bis(beta-aminoethyl ether)-N,N,NN′-tetraacetic acid(EGTA) was spun at 90,000×g for 5 minutes. The solution was then warmedto 37° C. for 2 minutes. Taxol was added in stepwise as 0.01, 0.1, and 1equivalents. The polymerization solution was placed onto a solutioncontaining 40% glycerol, 80 mM potassium HEPES (pH 6.8), 1 mM MgCl₂, and1 mM EGTA, and centrifuged at 90,000×g for approximately 50 minutes. Theresulting microtubule pellet was washed extensively with and resuspendedin resuspension buffer containing 80 mM potassium HEPES (pH 6.8), 1 mMMgCl₂, and 1 mM EGTA.

In Vitro NADH Enzyme Coupled ATPase: (Variation on Methods Described inCrevel, Lockhart, and Cross. J. Mol. Biol. (1997) 273, 160-170.)

For a brief description as the ATPase assay used in the present example,see FIG. 7.

A solution containing the microtubule resuspension buffer wassupplemented with 1 mM Mg-ATP, 100 uM nicotinamide adenine dinucleotide(NADH), 1 mM phosphoenol pyruvate, 5 ug/ml pyruvate kinase, 7.5 ug/mllactate dehydrogenase, 0.7 uM resuspended microtubules, and 0.5%dimethylsulfoxide (DMSO).

Next, serial dilutions of the compound 22C16 were prepared in thesupplemented microtubule resuspension buffer. Purified recombinant humanEg5 protein was added resulting in a final concentration of 50 uM. Thesubsequent fluorescent reaction was measured in NUNC black walled 384well plates at 340 nm using a Wallac plate reader.

FIGS. 8 and 9 show the inhibition of Eg5 ATPase activity by thebeta-carboline 22C16 described by the present invention and by monastrolrespectively. Based on these assays, the IC50 for 22C16 equals 1.1 uM.For monastrol, the IC50=5.3 uM. Therefore, based on the ATPase assaydescribed 22C16 is approximately 5 times more potent than monastrol inthe inhibition of the ATPase activity of the human kinesin Eg5.

EXAMPLE 3 Synthesis of Monastroline

The synthesis of monastroline (22C16) was performed via thePictet-Spengler reaction (Ber. 44:2030, 1911) Whaley and Govindachari.Org. Reaction. 6:74, 1951; Ungemach et al. J. Amer. Chem. Soc.102:6976-6984, 1980). Recent examples of the use of the Pictet-Spenglerreaction are provided by Rousseau and Dodd (J. Org. Chem. 63:2731-2737,1998) and by Leonard et al. (Tet. Lett. 38:3071-3074, 1997).

FIG. 10 depicts the synthetic reaction scheme for monastroline using thePictet-Spengler reaction. D,L-tryptophan (Sigma-Aldrich) was used as thestarting material. D,L-tryptophan was refluxed with3-hydroxylbenzaldehyde (Sigma-Aldrich) in methanol for approximately 6hours. The racemic beta-carboline was purified and refluxed withn-Bu-ICN in acetone for approximately 48 hours. Cis and trans isomers ofmonastroline were separated and purified by standard silica gelchromatography.

EXAMPLE 4 Synthesis of Monastroline Derivatives

FIG. 11 depicts the synthesis of phenyl derivatives of monastrolinewhich have a structure similar to monastroline and likely have similarbiological activity. In FIG. 11, monastroline and derivatives ofmonastroline may be conjugated to a solid support through the R groupwherein R is methyl, ethyl, n-butyl, or alpha-benzyl. Alternatively oradditionally, for conjugation to a resin or other solid support, R isC_(n)COOR′ where n is 1-20, and preferably equal to 1, 3, 5, 7, 9, 1 1,or 13, and where R′ is methyl, ethyl, n-butyl, or alpha-benzyl.

The synthesis of beta-carboline derivatives and further modifications ofthe beta-carboline core of monastroline is depicted in FIG. 12. Thesynthesis of derivatives having modifications, additions, andcombinations thereof to heteroatom functionality is depicted in FIG. 13.

Additionally syntheses of derivatives of monastroline are describedherein. The general synthetic scheme is depicted by the following:

Synthetic reactions involving Step A are readily appreciated by thoseskilled in the art. A representative reaction is shown below.

Reaction of Tryptophan derivatives (4) with aldehyde buiding blocks (5).

Syntheses of derivatives of monastroline starting with other derivativesof tryptophan are readily appreciated by those skilled in the art.Non-limiting examples are provided below.

Non-limiting examples of derivatives of monastroline resulting from thereaction depicted by Step A are provided below. The free acids can befurther reacted with an isocynanate. The carboxylic acids can beconverted to the amine (Tetrahedron. Lett. 39(11):1291-1294, 1998).

Syntheses involving the reaction depicted in Step B using isocyanatesare readily envisioned and appreciated by those skilled in the art. Anon-limiting example is provided below to further illustrate reactionsinvolving isocyanate in Step B.

Reaction of beta-carboline 4 with substituted isocyanates (2).

Non-limiting examples of derivatives resulting from reactions involvingStep B are provided below.

1. A compound having the structure:

wherein R_(1-4, 8-10), as valency and stability permit are eachindependently selected from the group consisting of substituted orunsubstituted, branched or unbranched alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heterocycle,heteroalkyl, OH, OR_(A), C(═O)R_(A), CO₂H, CO₂R_(A), CN, halogen, SH,SR_(A), SOR_(A), SO₂R_(A), NO₂, NH₂, NHR_(A), N(R_(A))₂, hydrogen andNHC(O)R_(A), wherein each occurrence of R_(A) as valency and stabilitypermit is independently selected from the group consisting ofsubstituted or unsubstituted, branched or unbranched alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,heteroalkyl, heterocycle, alkoxy, aryloxy, alkylthio, arylthio,heteroaryloxy, heteroarylthio, and hydrogen, and wherein R_(5-7, 11) asvalency and stability permit are each independently selected from thegroup consisting of substituted or unsubstituted, branched or unbranchedalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,heteroaryl, heterocycle, heteroalkyl, hydrogen, —(C═O)R_(B), and—(SO₂)R_(B), wherein each occurrence of R_(B) as valency and stabilitypermit is independently selected from the group consisting ofsubstituted or unsubstituted, branched or unbranched alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,heteroalkyl, heterocycle, alkoxy, aryloxy, alkylthio, arylthio,heteroaryloxy, heteroarylthio, hydrogen, and —(C═O)R_(C), wherein R_(C)as valency and stability permit is selected from the group consisting ofsubstituted or unsubstituted, branched or unbranched alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,heteroalkyl, heterocycle, alkoxy, aryloxy, alkylthio, arylthio,heteroaryloxy, heteroarylthio, and hydrogen.
 2. The compound of claim 1having the structure:

wherein X is H, O, halogen, CF₃, or carbon; and wherein R and R′ asvalency and stability permit are each independently selected from thegroup consisting of substituted or unsubstituted, branched or unbranchedalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,heteroaryl, heterocycle, heteroalkyl, hydrogen, —(C═O)R_(D), and—(SO₂)R_(D), wherein each occurrence of R_(C) as valency and stabilitypermit is independently selected from the group consisting ofsubstituted or unsubstituted, branched or unbranched alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,heteroalkyl, heterocycle, alkoxy, aryloxy, alkylthio, arylthio,heteroaryloxy, heteroarylthio, hydrogen, and —(C═O)R_(E), wherein R_(E)as valency and stability permit is selected from the group consisting ofsubstituted or unsubstituted, branched or unbranched alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,heteroalkyl, heterocycle, alkoxy, aryloxy, alkylthio, arylthio,heteroaryloxy, heteroarylthio, and hydrogen.
 3. (Canceled)
 4. Thecompound of claim 1 having the structure:

wherein Ar is phenyl, p-hydroxyphenyl, m-hydroxyphenyl, m-methoxyphenylor m-fluorophenyl and R is as valency and stability permit are eachindependently selected from the group consisting of substituted orunsubstituted, branched or unbranched alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heterocycle,heteroalkyl, hydrogen, —(C═O)R_(H), and —(SO₂)R_(H), wherein eachoccurrence of R_(H) as valency and stability permit is independentlyselected from the group consisting of substituted or unsubstituted,branched or unbranched alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalkyl, heterocycle,alkoxy, aryloxy, alkylthio, arylthio, heteroaryloxy, heteroarylthio,hydrogen, and —(C═O)R_(I), wherein R_(I) as valency and stability permitis selected from the group consisting of substituted or unsubstituted,branched or unbranched alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalkyl, heterocycle,alkoxy, aryloxy, alkylthio, arylthio, heteroaryloxy, heteroarylthio, andhydrogen.
 5. A pharmaceutical composition comprising at least one of thecompounds of claims 1, 2 or 4 and further comprising a pharmaceuticallyacceptable carrier.
 6. The composition of claim 5 wherein the compoundhas the structure:


7. (Canceled)
 8. (Canceled)
 9. The composition of claim 6 wherein thecompound has the stereochemistry:


10. A method of treating cancer in a subject comprising administering toa subject in need thereof a therapeutically effective amount of at leastone of any of the compounds of claims 1, 2 or 4 and a suitablepharmaceutically acceptable carrier.
 11. The method of claim 10 whereinthe compound has the structure:


12. The method of claim 11 wherein the compound has the stereochemistry: